Fungi as biotechnological allies: Exploring contributions of edible and medicinal mushrooms.

Edible and medicinal mushrooms possess excellent nutritional properties due to their incredible versatility in growing on different substrates and producing extracellular enzymes with a wide range of specificity. These features make them excellent candidates for various biotechnological applications. In this context, biotechnological applications using edible and medicinal mushrooms can focus on the bioprocessing of agro-industrial wastes, an economical and environmentally friendly strategy. This review, based on recent original research and scientific reviews, highlights the versatility and potential of mushrooms in terms of sustainability and efficiency. We emphasized the biotechnological applications of edible and medicinal mushrooms and their enzymes including food production with high nutraceutical value by enhancing the quality and flavor of food industry products. Other biotechnological applications addressed in this review were cosmeceutical and biomedical development using mushroom extracts with bioactive compounds; wood pulp pretreatment processes in the pulp and paper industry; bioethanol production; and bioremediation for decontaminating soils and polluted effluents. These applications explain how edible and medicinal mushrooms have gained significance in biotechnology over the years, opening new avenues for innovation. The current tendency to study edible and medicinal mushrooms has gained the attention of researchers because these are still less known organisms becoming an attractive and natural source of novel bioactive compounds that could be integrated into a circular model production.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems.

Heterologous expression of L-asparaginase (L-ASNase) has become an important area of research due to its clinical and food industry applications. This review provides a comprehensive overview of the molecular and metabolic strategies that can be used to optimize the expression of L-ASNase in heterologous systems. This article describes various approaches that have been employed to increase enzyme production, including the use of molecular tools, strain engineering, and in silico optimization. The review article highlights the critical role that rational design plays in achieving successful heterologous expression and underscores the challenges of large-scale production of L-ASNase, such as inadequate protein folding and the metabolic burden on host cells. Improved gene expression is shown to be achievable through the optimization of codon usage, synthetic promoters, transcription and translation regulation, and host strain improvement, among others. Additionally, this review provides a deep understanding of the enzymatic properties of L-ASNase and how this knowledge has been employed to enhance its properties and production. Finally, future trends in L-ASNase production, including the integration of CRISPR and machine learning tools are discussed. This work serves as a valuable resource for researchers looking to design effective heterologous expression systems for L-ASNase production as well as for enzymes production in general.

Regulation of lignocellulose degradation in microorganisms.

Microbial strategies for biomass deconstruction involve an incredible repertoire of enzymatic, structural, and regulatory proteins. From carbohydrate active enzymes to cellulosomes, bacteria, yeast, and filamentous fungi adapt their functional machinery to grow from alternative carbon sources such as lignocellulose and survive starvation. In that context, microbes must be able to sense, bind, degrade, and utilize lignin, cellulose, and hemicelluloses. Nature has developed specialized protein modules, RNA structures, and regulatory systems operating at a genomic, transcription, and translation level. This review briefly summarizes the main regulatory pathways involved in lignocellulose microbial degradation, including carbon catabolite repression; anti-sigma factors; regulatory RNA elements such as small RNAs, antisense RNA, RNA-binding proteins, and selective RNA processing and stabilization; and transcriptional regulators and unfolded protein response. Interplay with global regulators controlling pH response and nitrogen utilization is also revised.

New Papiliotrema laurentii UFV-1 strains with improved acetic acid tolerance selected by adaptive laboratory evolution.

The production of yeast oil from lignocellulosic biomasses is impaired by inhibitors formed during the pretreatment step, mainly acetic acid. Herein, we applied Adaptive Laboratory Evolution (ALE) to select three Acetic acid Tolerant Strains (ATS) of P. laurentii UFV-1. Different phenotypes emerged alongside evolution. The ATS II presented trade-offs in the absence of acetic acid, suggesting that it displays a specialized phenotype of tolerance to growth on organic acids. On the other hand, ATS I and ATS III presented phenotypes associated with the behavior of generalists. ATS I was considered the most promising evolved strain as it displayed the oleaginous phenotype in all conditions tested. Thus, we applied whole-genome sequencing to detect the mutations that emerged in this strain during the ALE. We found alterations in genes encoding proteins involved in different cellular functions, including multidrug resistance (MDR) transporters, energy metabolism, detoxification, coenzyme recycling, and cell envelope remodeling. To evaluate acetic acid stress responses, both parental and ATS I strains were cultivated in chemostat mode in the absence and presence of acetic acid. In contrast to ATS I, the parental strain presented alterations in the cell envelope and cell size under acetic acid stress conditions. Furthermore, the parental strain and the ATS I presented differences regarding acetic acid assimilation. Contrary to the parental strain, the ATS I displayed an increase in unsaturated fatty acid content irrespective of acetic acid stress, which might be related to improved tolerance to acetic acid. Altogether, these results provided insights into the mechanisms involved with the acetic acid tolerance displayed by ATS I and the responses of P. laurentii to this stressful condition.

Upstream and Downstream Bioprocessing in Enzyme Technology.

The development of biotransformation must integrate upstream and downstream processes. Upstream bioprocessing will influence downstream bioprocessing. It is essential to consider this because downstream processes can constitute the highest cost in bioprocessing. This review comprehensively overviews the most critical aspects of upstream and downstream bioprocessing in enzymatic biocatalysis. The main upstream processes discussed are enzyme production, enzyme immobilization methodologies, solvent selection, and statistical optimization methodologies. The main downstream processes reviewed in this work are biocatalyst recovery and product separation and purification. The correct selection and combination of upstream and downstream methodologies will allow the development of a sustainable and highly productive system.

Solid-state bioprocessing of sugarcane bagasse with Auricularia fuscosuccinea for phenolic compounds extraction.

Sugarcane bagasse is a natural source of phenolic compounds. However, these compounds are bound to lignocellulose components, reducing their ability to function as good antioxidants. These linkages are hydrolyzed by enzymes like ß-glucosidases, increasing free phenolics. Auricularia is a food-grade genus capable of producing ß-glucosidases. The aim of this work was (I) to determine naturally occurring species of Auricularia and (II) to obtain phenolic compounds through the solid-state bioprocessing of sugarcane bagasse. We have successfully isolated five strains that were assigned to the taxon A. fuscosuccinea. We determined ß-glucosidase activity by fluorescence plate assay of the five isolated strains and adjusted an optimal temperature for mycelial growth at 30 °C. A. fuscosuccinea LBM 243 was chosen for solid-state bioprocessing of sugarcane bagasse. ß-glucosidase activity (12.2 ± 0.62 U l-1) and protein content (51.58 ± 6.26 mg l-1) were highest on day 20 of culture. The maximum value of total phenolic content (507.5 ± 9.05 mg l-1) was obtained at day 20 and antioxidant capacity (34.44% ± 11.20) was highest at day 10, both in ethanolic extracts. The best performance of ethanol against methanol extraction in this work is highlighted considering ethanol to be a safe, efficient, and low-cost solvent.

Two-Step Purification of L-Asparaginase from Acrylaway® L

Abstract L-Asparaginase (L-ASNase) is a biopharmaceutical used for acute lymphoblastic leukaemia (ALL) treatment, dramatically increasing the patients' chance of cure. However, its production and distribution in developing countries were disrupted because of its low profitability, which caused great concern among patients. This study evaluates the feasibility of combining fractional precipitation and aqueous two-phase systems (ATPS) to purify L-ASNase from a low-grade product, commercially known as Acrylaway® L. The ATPS purification results were not particularly expressive compared to the two-step purification process composed of ethanol precipitation and gel filtration, which was able to recover the target molecule with a purification factor over 5 fold. Thus, we studied a purification process capable of manufacturing pharmaceutical grade L-ASNase from a commercially available low-grade raw material; however, improvements regarding its throughput must be achieved, and high purity is the first step to apply it as a new biopharmaceutical product. The proposed process could pose as a short-time solution to mitigate its shortage while a cost-effective production plant is being developed.

Microcarrier-based stem cell bioprocessing: GMP-grade culture challenges and future trends for regenerative medicine.

Recently, stem cell-based therapies have been proposed as an alternative for the treatment of many diseases. Stem cells (SCs) are well known for their capacity to preserve themselves, proliferate, and differentiate into multiple lineages. These characteristics allow stem cells to be a viable option for the treatment of diverse diseases. Traditional methodologies based on 2-dimensional culture techniques (T-flasks and Petri dishes) are simple and well standardized; however, they present disadvantages that limit the production of the cell yield required for regenerative medicine applications. Lately, microcarrier (MC)-based culture techniques have emerged as an attractive platform for expanding stem cells in suspension systems. Although the use of stem cell expansion on MCs has recently shown significant increase, their implementation for medical purposes is been hampered by bottlenecks in upstream and downstream processing. Therefore, there is an urgent need in the development of bioprocesses that simplify stem cell cultures under xeno-free conditions and detachment from MCs without diminishing their pluripotency and viability. A critical analysis of the factors that impact the up and downstream bioprocessing on MC-based stem cell cultures is presented in this review. This analysis aims to raise the awareness of the current drawbacks that limit MC-based stem cell bioprocessing in regenerative medicine and propose alternatives to overcome them.

Bioaccessibility and gut metabolism of phenolic compounds of breads added with green coffee infusion and enzymatically bioprocessed.

This study aimed at investigating two strategies to enhance the bioaccessibility of phenolic compounds from whole-wheat breads: enzymatic bioprocessing and addition of green coffee infusion. Although both strategies had a significant effect on increasing the contents of total soluble phenolic compounds in breads, the addition of green coffee infusion was much more relevant (19.1-fold) than enzymatic bioprocessing (1.8-fold). The phenolic compounds present as soluble forms were completely released from all breads' matrix already at the oral phase of digestion. While gastric digestion did not promote the release of insoluble phenolic compounds, intestinal conditions led to a slight release. All bread samples showed maximum phenolic compounds bioaccessibility after 4 h of gut fermentation. Upon the end of in vitro digestion and gut fermentation, the difference between the strategies was that enzymatic bioprocessing accelerated ferulic acid release, while the addition of green coffee infusion increased 10.4-fold the overall phenolic compounds bioaccessibility.

Biochemical and functional properties of wheat middlings bioprocessed by lactic acid bacteria.

The present study aimed to investigate the bioprocessing of wheat middlings with different lactic acid bacteria (LAB) in order to improve biological activities of this by-product of wheat flour production. The concentration of lactic acid, reducing sugars, and total phenolics, as well as antioxidant, antibrowning, antibacterial and prebiotic activities of fermented samples were analyzed. All LAB strains were capable to growth on wheat middlings, and pH decreased in the medium associated with lactic acid production during cultivation. Samples inoculated with Lactobacillus plantarum DSM20174 presented the maximum growth, lactic acid concentration above 2 mg/ml, and pH values around 3.8. The amount or reducing sugars decreased after 24 hr growth, except for maltose. Bioprocessed wheat middlings exhibited antioxidant, antibrowning, antibacterial, and prebiotic properties, related with the increase of total phenolic content. Highest values for antioxidant activities were obtained for L. plantarum and Streptococcus thermophilus strains, reaching values around 400 and 640 µM Trolox equivalents (TE) ml-1 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) assays, respectively. Bioprocessing techniques using LAB can be an interesting approach to improve the availability of compounds with health-promoting properties from lignocellulosic waste material. PRACTICAL APPLICATIONS: The processing of secondary products from wheat milling can represent an important benefit to the industry. Wheat middlings bioprocessed with LAB showed improved biological activities and may represent an interesting ingredient to be incorporated in food and feed formulations.

Laccases in Food Industry: Bioprocessing, Potential Industrial and Biotechnological Applications.

Laccase is a multi-copper oxidase that catalyzes the oxidation of one electron of a wide range of phenolic compounds. The enzyme is considered eco-friendly because it requires molecular oxygen as co-substrate for the catalysis and it yields water as the sole by-product. Laccase is commonly produced by fungi but also by some bacteria, insects and plants. Due it is capable of using a wide variety of phenolic and non-phenolic substrates, laccase has potential applications in the food, pharmaceutical and environmental industries; in addition, it has been used since many years in the bleaching of paper pulp. Fungal laccases are mainly extracellular enzyme that can be recovered from the residual compost of industrial production of edible mushrooms as Agaricus bisporus and Pleurotus ostreatus. It has also been isolated from microorganisms present in wastewater. The great potential of laccase lies in its ability to oxidize lignin, one component of lignocellulosic materials, this feature can be widely exploited on the pretreatment for agro-food wastes valorization. Laccase is one of the enzymes that fits very well in the circular economy concept, this concept has more benefits over linear economy; based on "reduce-reuse-recycle" theory. Currently, biorefinery processes are booming due to the need to generate clean biofuels that do not come from oil. In that sense, laccase is capable of degrading lignocellulosic materials that serve as raw material in these processes, so the enzyme's potential is evident. This review will critically describe the production sources of laccase as by-product from food industry, bioprocessing of food industry by-products using laccase, and its application in food industry.

A New Salt-Tolerant Thermostable Cellulase from a Marine Bacillus sp. Strain.

A salt-tolerant cellulase secreted by a marine Bacillus sp. SR22 strain with wide resistance to temperature and pH was purified and characterized. Its approximate mass was 37 kDa. The endoglucanase, named as Bc22Cel, was purified by ammonium sulfate precipitation, gel filtration chromatography, and extraction from the gel after non-reducing sodium dodecyl sufate-polyacrylamide gel electrophoresis. The optimal pH value and temperature of Bc22Cel were 6.5 and 60°C, respectively. The purified Bc22Cel showed a considerable halophilic property, being able to maintain more than 70% of residual activity even when pre-incubated with 1.5 M NaCl for 1 h. Kinetic analysis of the purified enzyme showed the Km and Vmax to be 0.704 mg/ml and 29.85 µmol·ml-1·min-1, respectively. Taken together, the present data indicate Bc22Cel as a potential and useful candidate for industrial applications, such as the bioconversion of sugarcane bagasse to its derivatives.

Microbial dynamics in anaerobic digestion reactors for treating organic urban residues during the start-up process.

Anaerobic digestion of organic residues offers economic benefits via biogas production, still methane (CH4 ) yield relies on the development of a robust microbial consortia for adequate substrate degradation, among other factors. In this study, we monitor biogas production and changes in the microbial community composition in two semi-continuous stirred tank reactors during the setting process under mesophilic conditions (35°C) using a 16S rDNA high-throughput sequencing method. Reactors were initially inoculated with anaerobic granular sludge from a brewery wastewater treatment plant, and gradually fed organic urban residues (4·0 kg VS m-3  day-1 ) . The inocula and biomass samples showed changes related to adaptations of the community to urban organic wastes including a higher relative proportion of Clostridiales, with Ruminococcus spp. and Syntrophomonas spp. as recurrent species. Candidatus Cloacamonas spp. (Spirochaetes) also increased from ~2·2% in the inoculum to >10% in the reactor biomass. The new community consolidated the cellulose degradation and the propionate and amino acids fermentation processes. Acetoclastic methanogens were more abundant in the reactor, where Methanosaeta spp. was found as a key player. This study demonstrates a successful use of brewery treatment plant granular sludge to obtain a robust consortium for methane production from urban organic solid waste in Mexico. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the selection of relevant bacteria and archaea in anaerobic digesters inoculated with anaerobic granular sludge from a brewery wastewater treatment plant. Generally, these sludge granules are used to inoculate reactors digesting organic urban wastes. Though, it is still not clearly understood how micro-organisms respond to substrate variations during the reactor start-up process. After feeding two reactors with organic urban residues, it was found that a broader potential for cellulose degradation was developed including Bacteroidetes, Firmicutes and Spirochaetes. These results clarify the bacterial processes behind new reactors establishment for treating organic wastes in urban areas.

Blood Sampling and Preparation Procedures for Proteomic Biomarker Studies of Psychiatric Disorders.

A major challenge in proteomic biomarker discovery and validation for psychiatric diseases is the inherent biological complexity underlying these conditions. There are also many technical issues which hinder this process such as the lack of standardization in sampling, processing and storage of bio-samples in preclinical and clinical settings. This chapter describes a reproducible procedure for sampling blood serum and plasma that is specifically designed for maximizing data quality output in two-dimensional gel electrophoresis, multiplex immunoassay and mass spectrometry profiling studies.

Bioprocessing of common beans in diets for tilapia: in vivo digestibility and antinutritional factors.

BACKGROUND: Bioprocessing of ingredients by solid-state fermentation is a low-cost technique for preparing diets. It is performed by adding microorganisms such as Rhizopus oligosporus to bean grains, achieving minimal degradation of nutrients and a significant improvement in digestibility. In particular, fermentation induces favorable changes in beans by reducing enzyme inhibitors, such as phytates and tannins. RESULTS: Fermentation significantly (P < 0.05) increased the protein content and digestibility of dry matter and protein compared with whole bean grains, and decreased the content of lipids, ash and phytic acid. Hardening did not have a significant (P > 0.05) effect on the chemical content of beans and digestibility of diets. The dehulled bean meal significantly (P < 0.05) increased protein and lipid content and digestibility of dry matter and protein of beans, and decreased fiber, ash and tannin content. The chemical content of beans and digestibility of ingredients compare favorably with those reported by other authors, indicating the benefits of fermentation and dehulling. CONCLUSION: We concluded that bean meal obtained from fermentation or dehulling represents a low-cost alternative for diets for tilapia. © 2017 Society of Chemical Industry.

Blood Bio-Sampling Procedures for Multiplex Biomarkers Studies.

A major challenge in single or panel of biomarker discovery and validation is the inherent biological complexity underlying disease heterogeneity and inconsistent responses to treatment. Moreover, the lack of standardization in the sampling, processing, and storage of biological fluids such as plasma and serum disrupts the discovery and validation of blood-based biomarker tests in preclinical and clinical settings. This chapter presents a reproducible sample collection and handling procedure that aims to enhance analyte stability and ensure compatibility with the corresponding multiplex biomarker profiling platforms. The importance of defining bio-sample acquisition and processing, study design, and profiling platform guidelines for blood-based biomarker measurements is paramount for the success of personalized healthcare strategy and development of companion diagnostics.

Progress in bacterial cellulose matrices for biotechnological applications.

Bacterial cellulose (BC) is an extracellular polymer produced by many microorganisms. The Komagataeibacter genus is the best producer using semi-synthetic media and agricultural wastes. The main advantages of BC are the nanoporous structure, high water content and free hydroxyl groups. Modification of BC can be made by two strategies: in-situ, during the BC production, and ex-situ after BC purification. In bioprocesses, multilayer BC nanocomposites can contain biocatalysts designed to be suitable for outside to inside cell activities. These nanocomposites biocatalysts can (i) increase productivity in bioreactors and bioprocessing, (ii) provide cell activities does not possess without DNA cloning and (iii) provide novel nano-carriers for cell inside activity and bioprocessing. In nanomedicine, BC matrices containing therapeutic molecules can be used for pathologies like skin burns, and implantable therapeutic devices. In nanoelectronics, semiconductors BC-based using salts and synthetic polymers brings novel films showing excellent optical and photochemical properties.

Effects of agitation and exogenous H2 on bioconversion of sugarcane bagasse into ethanol by Clostridium thermocellum ATCC 27405

Background: The production of ethanol by a Consolidated Bioprocessing (CBP) strategy, which simultaneously combines cellulase production, lignocellulosic biomass hydrolysis and fermentation of released sugars to ethanol in one bioreactor, is a promising technology for cost reduction in the biological processing of biomass, specially using agroindustrial residues. Clostridium thermocellum is an anaerobic, thermophilic, strictly fermentative gram positive bacterium that meets all the requirements for CBP. Results: Ethanol concentration obtained in the non-stirred fermentation process in flasks with raw bagasse was two times greater than that in the stirred system. The results observed using a pretreated sugarcane bagasse in non-stirred flasks regarding ethanol concentration, were slightly lower than with raw bagasse. The sparging of exogenous H2 into the medium at atmospheric pressure inside the bioreactor showed to be unfavourable to achieve higher ethanol yields. Conclusions: The strain investigated is a promising candidate for thermophilic fermentative ethanol production from dried ground raw sugarcane bagasse in a CBP strategy, although the alcohol concentrations need to be further improved. In future studies, it is recommended to investigate different modes of operation of the fermentation process, including pressurized conditions, as well as to use wet raw sugarcane bagasse aiming to achieve additional improvement in ethanol production and to reduce the costs of the process.

Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae.

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.